• Journal of Embryo Transfer
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• v.20 no.1
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• pp.1-8
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• 2005

This experiment was carried out to investigate the effects of saccharide in the lactose-egg yolk(LEY) extender for freezing of boar semen on the viability, normal acrosome, fertilizable of in vitro or in vivo oocyte after thawed. Normal acrosome post-thawed spermatozoa was higher when increasing of glucose concentration in LEY extender with 3 or $4\%$ glycerol, but viability was not significant. Viability of the post-thawed spermatozoa was higher when fructose or fructose and glucose were added to LEY extender with $3\%$ glycerol than glucose and sucrose or fructose, glucose and sucrose(P<0.05). Rate of normal acrosome of post thawed spermatozoa was higher when both fructose and glucose$(81.4{\pm}2.3\%)$ were added to the LEY extender than saccharide not added$(41.6\pm0.6\%)$ to it(P<0.001). The percentage of fertilization, cleavage and development to blastocyst of oocytes fertilized with post-thawed spermatozoa from freezing by LEY extender were $70.8\~80.7\%$, $44.6\~45.7$ and $13.6\~16.0\%$, respectively. Conception rate by artificial insemination with frozen boa. semen was higher$(83.1{\pm}0.3\%)$ than commercial frozen semen from SGI company$(50.0{\pm}0.1\%,\;P<0.05)$, but litter size were no significant differences between frozen by LEY extender$(9.4{\pm}1.7\~10.4{\pm}0.7head/sow)$ and SGI semen$(8.0{\pm}1.1 head/sow)$.

• KSBB Journal
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• v.16 no.1
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• pp.41-47
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• 2001

H. pylori is known to be a key pathogen of chronic gastric and duodenal ulcers. Bacterial adhesion to hosts is an essential step for bacterial infection and the inhibition of this adhesion provides a possible method for the treatment of the infection. The inhibitory effect of antibody lgY, produced from immunized hens with H. pylori antigen, was studied in vitro. The inhibition of H. pylori adhesion to AGS was as high as 90% using 0.5mg/ml of lgY, and almost 80% of the detachmentwas also achieved. The inhibitory effect of adhesion-inhibition candidates was investigated. Additives in combination with lgY increased the adhesion-inhibiting effect by about 30-50%. However, the adhesion molecules of H. pylori were varied and complex, therefore the further studies are necessary to develop an adhesion inhibitor and effective enough to be employed for the treatment of H.pylori, in vivo.

• Journal of Embryo Transfer
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• v.25 no.3
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• pp.195-199
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• 2010

This studies were conducted to investigate the survival rate of frozen-thawed spermatozoa of Jindo Dog by monosaccharide and freezing rates. Experimental animals were prepared 12 males within 1~8 year's old and collected once in a couple of weeks by digital manuplation methods. Collected semen was diluted 1:1 with Tris-egg yolk extender and added 4, 6 or 8% of glycerol and none, 4 mM glucose or 4 mM fructose as cryoprotectant and was equilibrated for 2 hrs in $4^{\circ}C$. In monosaccharide groups, the freezing rate was 5 cm-5 min. above $LN_2$. The survival rates without monosaccharide were $50.7{\pm}19.0%$, $58.6{\pm}18.0%$, $40.0{\pm}10.0%$ in 4, 6 or 8% glycerol, respectively. In addition of glucose, the survival rates were $43.1{\pm}14.7%$, $38.1{\pm}16.5%$, $33.3{\pm}4.0%$ in 4, 6 or 8% glycerol, respectively and in fructose, were $47.9{\pm}21.1%$, $61.3{\pm}6.2%$, $34.3{\pm}12.6%$ in 4, 6 or 8% glycerol, respectively. There showed significantly different between glycerol groups and monosaccharides groups (p<0.05). The survival rates of freezing rate in 5 cm-5 min. group was $64.5{\pm}15.8%$, $51.9{\pm}27.6%$, $29.7{\pm}24.8%$ and in 10 cm-10 min. group was $62.5{\pm}20.3%$, $64.9{\pm}23.6%$, $34.5{\pm}27.4%$ in 4, 6 or 8% glycerol, respectively. There were significantly different between freezing rates (p<0.05). These results suggest that the addition of fructose with 6%-glycerol and slow freezing improve the survival of frozen-thawed sperm in Jindo Dog.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.185-194
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• 2018

The aim of this study was performed to evaluate the effects of ice-binding protein from the arctic yeast Leucosporidium (LeIBP) supplementation on cryopreservation of boar sperm. The collected semen was diluted ($1.5{\times}10^8/ml$) in lactose egg yolk (LEY) and cooled at $5^{\circ}C$ for 3 h. The cooled semen was then diluted ($1{\times}10^8/ml$) in LeIBP containing LEY with 9% glycerol and maintained at $5^{\circ}C$ for 30 min. The semen was divided into six experimental groups (control, 0.001, 0.005, 0.01, 0.05 and 0.1 mg/ml of LeIBP). The straws were kept on above the liquid nitrogen ($LN_2$) vapors for 20 minutes and then plunged into $LN_2$. After thawing, computer-assisted sperm analysis was used for sperm motility and flow cytometry was performed to assess the viability, acrosome integrity (FITC-PSA/PI), ROS (DCF/PI), lipid peroxidation (BODIPY C11/PI) and apoptosis (Annexin V/PI), respectively. No significant responses were observed for sperm motility. However, sperm viability was significantly increased on 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). In addition, acrosome integrity was significantly increases LeIBP groups (P < 0.05) and both ROS and lipid peroxidation level were lower in all LeIBP groups than those of control (P < 0.05). On the other hand, a significant higher apoptosis rate was observed in 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). It can be assumed that a supplementation of LeIBP in boar sperm freezing extender is an effective method to increase the sperm qualities after cryopreservation.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.271-274
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• 2010

This study was conducted to investigate the survival rate of frozen-thawed spermatozoa in equine by glycerol concentration and freezing speed Two stallions (1 Thoroughbred-13 year old and 1 Arab-7 year old) bred in Korea Racing authority was examined for 1 times in a couple of weeks. Semen was collected by condom method standing heated mare and were centrifuged 650 g for 15 min. and isolated the seminal plasma. Thick fraction of semen was diluted EDTA-Lactose-egg yolk diluents to 1:1 and contained in 0.5 ml straw as $6{\sim}14{\times}10^7\;cells/ml$. Final concentrations of glycerol were 3, 5 and 7% in cryopreseved diluents and added 4 times for 2 hours equilibration. For the freezing, equilibrated straws were located 3 or 5 em above $LN_2$ gas for 5 or 10 min. Survival rates of pre-frozen sperm were $65.0{\pm}13.2%$, $68.3{\pm}10.4%$, $66.7{\pm}11.5%$ and post-frozen were $53.3{\pm}23.1%$, $45.0{\pm}15.0%$, $50.0{\pm}18.0%$ in 3, 5, 7% glycerol concentration, respectively. There was no difference between glycerol concentrations. Survival rates of frozen-thawed sperm on freezing speed were $36.7{\pm}10.4%$, $40.0{\pm}7.1%$, $30.0{\pm}13.2%$ at 3 cm-5 min and $33.3{\pm}11.5%$, $31.7{\pm}2.9%$, $21.7{\pm}10.4%$ at 3 cm-10 min in 3, 5, 7% glycerol concentration, respectively. Survival rates of frozen-thawed sperm on freezing speed were $43.3{\pm}15.3%$, $32.0{\pm}17.9%$, $22.3{\pm}15.7%$ at 5cm-5 min and were $47.5{\pm}15.0%$, $43.3{\pm}12.6%$, $48.3{\pm}15.3%$ at 5cm-10 min in 3, 5, 7% glycerol concentration, respectively. There were significantly different between groups (p<0.05). These results suggest that glycerol concentration did not affect cryopreservation of stallion semen within 3~7% but freezing speed affects. In our experiment, the best cryopreservation condition was at 5 cm above $LN_2$ gas for 10 min for pre-freezing and 7% of glycerol concentration. These results lead to commercial AI with frozen-thawed stallion semen.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.55-60
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• 2011

The objective of this study was carried out to evaluate the efficiency of sperm collection methods on the post-thaw viability of cat semen. The cat semen was collected by artificial virginal (AV) and electronic ejaculate (EE) methods. The composition of semen extender was consisted of Tris-buffer supplemented with 20% egg yolk and 1% P/S antibiotics in Ext I, and more added 8% glycerol, 1.0% Equex STM paste of total volume in Ext II. The collected semen was adjusted the concentration and then diluted in Ext I for optimal concentration. The diluted semen was cooling to $5^{\circ}C$ temperature in refrigerator for at least 2 hrs and then diluted stepwise with Ext II for at least 1 hrs. After an equilibration for 1 hrs, the cooled semen was packaged in 0.5 ml straw and then freezing on the $LN_2$ vapor over 5 cm above from $LN_2$ and then immersed directly in $LN_2$ for cryopreservation. The frozen semen was thawed in $38^{\circ}C$ water for 15 sec and then evaluated the motility, viability, and morphology. Post-thaw semen were calculated the motility by SMI (sperm motility index). The live-dead sperm was evaluated by Eosin-B and morphological evaluation was by Diff-quik kit staining. The post-thaw concentration ($89{\times}10^6$ /ml vs. $128{\times}10^6$ /ml), viability ($22.6{\pm}10.6%$ vs. $37.1{\pm}26.1%$), morphological normality ($27.0{\pm}50.2%$ vs. $45.6{\pm}123.0%$) of EE and AV groups were not significant different, but the post-thaw motility was significant lower in EE than that in AV group ($53.1{\pm}3.6$ vs. $73.6{\pm}5.7$) (p<0.05). In conclusion, semen collection methods did not significant different between EE and AV groups except of post-thaw motility and so both semen collection methods could be applied in feline semen collection methods.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.61-65
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• 2011

The objective of this study was to examine the effect of various discontinuous Percoll washing conditions on motile sperm recovery rate and motion kinematics. Frozen semen samples from 3 bulls (0.5 ml plastic straws, 6% glycerol in egg yolk-Tris-glycerol extender) were thawed in $37^{\circ}C$ water bath for 1 min. After thawing, the mixed semen samples were randomly allocated to 12 treatment groups. Briefly, the spermatozoa were centrifuged for three different time lengths (10, 20, and 30 min) at two gravities ($300{\times}g$ and $700{\times}g$) through two concentrations of discontinuous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll and 2 ml 90%: 2 ml 45% Percoll to remove extender, debris, and dead spermatozoa. Motile sperm recovery rate and motion kinematics were evaluated by computer assisted sperm analyzer using Makler counting chamber. Sperm motility (%) and motile sperm recovery rate showed similar pattern in all treatment groups. However, sperm motility (%) and motile sperm recovery rate were highest at $700{\times}g$ for 30 min through a discontionous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll. There were no significant differences in motion kinematics after various Percoll washings. These results suggest that force of centrifugation, centrifugation time, and Percoll volume significantly affect motile sperm recovery rate.

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.163-169
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• 2014

The objective of this study was to evaluate the characteristics of Korean Native Cattle sperm frozen-thawed with L-cysteine and/or catalase. The semen from bulls was collected by the artificial vagina method, and Triladyl containing 20% egg-yolk and/or L-cysteine (L), catalase (C) and L-cysteine + catalase was added to the diluted semen for cryopreservation. The results showed that sperm viability was significantly higher in the L-cysteine + catalase ($69.49{\pm}3.16%$) group than in the control ($60.5{\pm}3.94%$) group (p<0.05). Acrosome damage was significantly lower in the L-cysteine ($17.12{\pm}1.08%$) group than in the control ($21.46{\pm}1.14%$), catalase ($20.54{\pm}0.76%$), and L-cysteine + catalase ($19.29{\pm}0.65%$) groups (p<0.05). In addition, the level of intact mitochondria in the spermatozoa was significantly higher in the L-cysteine ($58.65{\pm}1.39%$) group than in the control ($50.63{\pm}2.37%$) group (p<0.05). The hydrogen peroxide level in the frozen-thawed sperm was significantly lower in the L-cysteine ($3.74{\pm}1.66%$), catalase ($4.65{\pm}1.87%$), and L-cysteine + catalase ($8.11{\pm}2.15%$) groups than in the control ($13.22{\pm}1.6%$) group (p<0.05). The glutathione level was significantly higher in the L-cysteine ($1.33{\pm}0.03%$) group than in the control ($1.08{\pm}0.06%$), catalase ($1.05{\pm}0.02%$) and L-cysteine + catalase ($1.11{\pm}0.03%$) groups (p<0.05). In conclusion, L-cysteine and catalase could protect the membrane of Korean Native Cattle sperm from damage during sperm cryopreservation. Especially, L-cysteine was more effective for keeping acrosomes and mitochondria intactness during sperm cryopreservation.

• Korean Journal of Food Science and Technology
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• v.36 no.1
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• pp.134-140
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• 2004

Optimal extraction method for antioxidant compounds from coat of grape dreg was established. Extracts were prepared with ethanol solutions containing phosphoric, formic, acetic, HCl, TFA, and citric acids. Antioxidant compounds of grape coat were determined by HPLC analysis and evaluated for antioxidizing effects using in vitro model system. Peonidin-3-glucoside content was the highest in 0.1% HCl-added ethanol extract. The extract prepared from pure ethanol without organic acids showed the highest content of cyanidin-3-glucoside among samples tested. Resveratrol and quercetin contents, the most important antioxidants, were highest in 0,1% HCl-added extract. Electron-donating ability was high in 0.1% acetic acid-added extract. SOD-like activities were 95.08% and 94.39% in 0.1% formic and 0.1% citric acid extracts, respectively. Inhibitory effects on peroxidation of egg yolk lecithin were observed in phosphoric (60.51%), citric (59,27%), and formic acid (56,77%) extracts. Hydrogen radical-scavenging activity was 59.47% in 0,1% HCl extract. Results suggest addition of 0.1% HCl in ethanol solution affords the highest yield in antioxidant compounds and antioxidant activities.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.12
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• pp.1721-1727
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• 2008

Four experiments were conducted to study the following: i) the influence of different concentrations of sucrose (0.15, 0.3 and 0.5 M with osmolality of 308, 500 and 760 mOsm/kg, respectively) in diluents and control diluent (370 mOsm/kg) on intensity of motility and progressive motility of goat sperm without rehydration and freezing step in four incubation periods (0, 0.5, 2 and 4 h after dilution); ii) the influence of gradual dilution (in 3 steps) on improvements in ascertained results of the first experiment; iii) cryoprotective effects of different concentrations of sucrose (0.15, 0.22, 0.29 and 0.37 M with osmolality of 450, 560, 740 and 920 mOsm/kg, respectively) plus 7% glycerol and 20% egg yolk in basic diluent (Tris-Citric acid-Fructose) and iv) the effect of two concentrations of sucrose (0.15 and 0.22 M) with and without glycerol (7%). In experiment 1, we obtained better results for control diluent, 0.15 and 0.3 M sucrose supplemented diluents with 0 and 0.5 h incubation periods. In experiment 2, apart from a slight improvement, similar tendencies to experiment 1 were observed. In experiment 3, we obtained the best result for diluent with 0.22 M sucrose with regard to intensity of motility, progressive motility, live sperm and normal acrosomes ($40{\pm}4%$, $3.1{\pm}0.2$, $37{\pm}4%$ and $37{\pm}4%$, repectively). In experiment 4, we obtained the best result for diluent with 0.22 M sucrose plus 7% glycerol in regard to intensity of motility, progressive motility and live sperm ($39{\pm}3%$, $3.6{\pm}0.4$ and $41{\pm}4%$, respectively). The characteristic normal acrosomes in diluents without glycerol, i.e. diluents with 0.15 and 0.22 M sucrose showed better results ($39{\pm}8$ and $42{\pm}6%$ respectively). With regard to the release of hyaluronidase enzyme there were no significant differences between diluents (p>0.05). The results of the diluents with 0.15 and 0.22 M sucrose without glycerol were slightly lower than those with glycerol ($69{\pm}11$ and $70{\pm}11$ vs. $72{\pm}11$ and $70{\pm}11{\times}120{\times}10^6$ units $ml^{-1}$, respectively). In conclusion, the use of concentrated sucrose solutions showed that goat sperm can tolerate osmolality up to 560 mOsm (0.22 M) in the freezing period. In addition, glycerol proved to be a necessary cryoprotective agent in the cryopreservation of goat sperm, particularly for intensity of motility, progressive motility and live sperm.